Journal of Experimental Marine Biology and Ecology 345 (2007) 110 – 118

www.elsevier.com/locate/jembe

Glutathione protection against dive-associated ischemia/reperfusion

in ringed seal tissues
José Pablo Vázquez-Medina a , Tania Zenteno-Savín a,⁎, Robert Elsner b
a
Planeación Ambiental y Conservación, Centro de Investigaciones Biológicas del Noroeste, S.C. Mar Bermejo 195,
Playa Palo de Santa Rita, C.P. 23090, La Paz, B. C. S., México
b
Institute of Marine Science, University of Alaska Fairbanks, Fairbanks, AK, 99775, USA
Received 3 January 2007; received in revised form 13 February 2007; accepted 15 February 2007

Abstract

Ischemia/reperfusion is a potentially hazardous condition that increases reactive oxygen species (ROS) production and
oxidative damage. Seals of the phocid family experience repetitive episodes of ischemia/reperfusion during and after a dive as a
consequence of preferential distribution of blood flow to the central nervous system and reduction or elimination of perfusion in
most vascular beds. Previous studies showed that ROS production is higher in ringed seal than in domestic pig tissues as a direct
consequence of the ischemia/reperfusion associated with the diving response; however, oxidative damage is not related to this high
ROS production. Apparently, antioxidant enzyme activities participate in the antioxidant protection in ringed seal tissues. In the
present study we addressed the potential antioxidant protection of the glutathione system against dive-induced ischemia/reperfusion
in ringed seal tissues. Total glutathione (GSH-Eq = GSH + 2GSSG), glutathione (GSH) and glutathione disulfide (GSSG), the ratio
GSSG:GSH-Eq, the activities of the enzymes glutathione disulfide reductase (GR) and glucose-6-phosphate dehydrogenase
(G6PDH), as well as lipid peroxidation (TBARS) and carbonyl proteins, were measured in ringed seal and domestic pig heart,
kidney, liver, lung and muscle samples. In heart, kidney, lung and muscle GSH-Eq and GSH content was higher in seal than in pig
( p b 0.05). GSSG content was higher in seal than in pig heart kidney, liver and muscle ( p b 0.05). GR and G6PDH activities were
higher in all seal than in pig tissues ( p b 0.05). GSSG:GSH-Eq ratio was higher in pig than in seal heart, and lung ( p b 0.05).
TBARS content was higher in pig than in seal lung ( p b 0.05). Higher content of carbonyl proteins was present in pig than in seal
heart, kidney, liver and muscle ( p b 0.05). These results suggest that the glutathione levels and the activity of enzymes involved in
its recycling are efficient mechanisms that ameliorate protein and lipid oxidative damage and protect ringed seal tissues against
dive-induced ischemia/reperfusion.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Antioxidants; Diving; Glutathione; Ischemia/reperfusion; Seals

1. Introduction

Ischemia/reperfusion is a physiological condition that

⁎ Corresponding author. Programa de Planeación Ambiental y
raises the production of reactive oxygen species (ROS)
Conservación, Centro de Investigaciones Biológicas del Noroeste, S.C.
Mar Bermejo 195, Playa Palo Santa Rita, La Paz, Baja California Sur, CP and the potential for oxidative damage. During ischemia,
23090 - México. Tel.: +52 612 123 8502; fax: +52 612 125 3625. ATP depletion results in purine nucleotide accumulation
E-mail address: tzenteno04@cibnor.mx (T. Zenteno-Savín). and proteolytic conversion of xanthine dehydrogenase
0022-0981/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jembe.2007.02.003
 J.P. Vázquez-Medina et al. / Journal of Experimental Marine Biology and Ecology 345 (2007) 110–118
(XDH) to xanthine oxidase (XO). During reperfusion,
XO is able to reduce purine nucleotides in presence of
U
oxygen (O2) and to generate superoxide radical (O2−),
U
hydrogen peroxide (H2O2) and hydroxyl radical (HO ).
U− U
O2 and HO can react with proteins, lipids and nucleic
acids causing cellular injury (McCord and Fridovich,
1969; Kuppusamy and Zweier, 1989; Thompson-Gor-
man and Zweier, 1990; Halliwell and Gutterdige, 1999).
Mechanisms that prevent deleterious effects of ROS
in animal cells include antioxidant enzymes such as
superoxide dismutases (Cu,Zn-SOD, Mn-SOD), cata-
lase (CAT), selenium-dependent glutathione peroxidases
(GPx) and glutathione-S-transferases (GST) (Halliwell
and Gutterdige, 1999). Antioxidant enzymes remove or
transform ROS into less toxic metabolites; SOD cata-
U
lyzes O2− dismutation into H2O2, CAT and GPx remove
H2O2, and GST detoxifies xenobiotics and oxidized
products of ROS reactions (Halliwell and Gutterdige,
1999). GPx and GST consume glutathione (GSH) in
their catalytic reactions yielding glutathione disulfide
(GSSG) (Prohaska, 1980). GSH is the most abundant
non-protein tri-peptide thiol present in animal cells; it is
formed by cystein, glicyne and glutamic acid residues
and resists intracellular degradation by peptidases.
U
GSH participates in HO and singlet oxygen scavenging
and is recycled from GSSG by the action of the enzyme
glutathione disulfide reductase (GR) at the expense of
NADPH, preventing GSH loss (Halliwell and Gutter-
dige, 1999; Sies, 1999). The GSSG/2GSH ratio is used
as an indicator of the redox status in the cell (Kosower
and Kosower, 1978; Meister and Anderson, 1983).
The dive response in seals involves heart rate reduc-
tion (bradycardia) and preferential distribution of oxy-
genated blood flow to the heart and brain by a widespread
peripheral vasoconstriction which results in selective
ischemia in the most hypoxia-tolerant tissues during a
dive. The first breath after a dive restores the oxygenated
blood flow to all tissues (reperfusion) raising the potential
for the production of ROS (Gooden and Elsner, 1985;
Elsner et al., 1995, 1998; Elsner, 1999). Phocid seals
tolerate repetitive cycles of ischemia/reperfusion that
results in ROS production but apparently not in oxidative
damage (Zenteno-Savin and Elsner, 1998, 2000; Zenteno-
Savin et al., 2002). Antioxidant enzymes are actively
involved in seal tissue protection (Vazquez-Medina et al.,
2006). Until now, no evidence on the participation of
glutathione as an antioxidant against ROS in diving
seal tissues is available. The findings of Murphy and
Hochachka (1981) suggest unusual changes in glutathi-
one levels in Weddell seal blood during and after forced
dives. In this study, we address the role of glutathione
and its recycling in protecting ringed seal tissues against
111
dive-induced ischemia/reperfusion by comparing the
glutathione levels and the activity of the enzymes
involved in its recycling between ringed seal and domestic
pig tissue samples. We recognize the limitations of com-
parative studies based on only two species. The choice
of pig tissues for comparisons is based on similarities
of pigs to seals in metabolic scope and body fat con-
tents (Irving, 1956; Irving et al., 1956) and clear dif-
ferences in the diving adaptations of the two species
(Elsner, 1999).
2. Materials and methods
2.1. Sample collection
Approximately 5 g of heart, kidney, liver, lung and
muscle tissue samples from 11 ringed seals (Phoca
hispida hispida Schreber, 1775) of both sexes (7 males
and 4 females, average body mass 31.98 ± 3.47 kg) were
obtained incidental to subsistence hunting with the
collaboration of the North Slope Borough, the Alaska
Department of Wildlife Management and Inupiat Eski-
mo hunters, near Barrow, Alaska, U.S.A. For compar-
ative purposes, tissue samples from 10 pigs (Sus scrofa
domesticus Linnaeus, 1758) (5 males and 5 females,
average body mass 73.40 ± 1.10 kg) were obtained at a
local slaughter-house in La Paz, Baja California Sur,
Mexico. All tissue samples were excised immediately
following death of the animals and frozen by immersion
in liquid nitrogen for transportation and subsequently
stored at − 80 °C.
2.2. Reagents
All chemicals were purchased from Sigma–Aldrich
Chemical Co. (St. Louis, MO, USA.), except 2-
vinylpiridine (2VP), which was purchased from Fluka
(Hanover, Germany). Protein assay kits were purchased
from Biorad Laboratories (Hercules, CA, USA).
2.3. Glutathione assay
Frozen tissue samples were homogenized (1:10 w/v)
in ice-cold sulfosalicylic acid (5%) previously bubbled
with nitrogen gas for 10 min. Tissue extracts were
bubbled with nitrogen gas for 10 s and centrifuged at
19,000 ×g at 4 °C for 5 min. Supernatants were kept and
used immediately to measure total glutathione (GSH-
Eq = GSH + 2GSSG) levels. GSH-Eq were determined
by following the rate of reduction of 5,5′-dithiobis-2-
nitrobenzoic acid (DTNB). Briefly, in a cuvette, po-
tassium phosphate buffer (KPi 125 mM, pH 7.2; EDTA
112 J.P. Vázquez-Medina et al. / Journal of Experimental Marine Biology and Ecology 345 (2007) 110–118

6 mM), NADPH (0.3 mM), GR (50 U/mL), DTNB
(6 mM), deionized water and 50 μL of sample were
mixed. Change in absorbance per minute at 412 nm
(ΔA412) was measured and GSH-Eq concentration in
each sample was calculated from a standard curve (0–
4 mM) (Hermes-Lima and Storey, 1995, 1996).
Griffith's method with modifications (Griffith, 1980;
Hermes-Lima and Storey, 1993; Ramos-Vasconcelos
and Hermes-Lima, 2003) was used to quantify GSSG
concentration. Sample extracts were mixed with 2VP
(0.5 M), KPi buffer (0.5 M, pH 7.0) and adjusted to pH
7.0 with NaOH (1 M). Samples were incubated in the
dark for 1 h. Then, deionized water, 150 μL of sample,
NADPH (0.3 mM), GR (50 U/mL) and DTNB (6 mM)
were mixed in a cuvette and absorbance was measured
at 412 nm during 130 s. GSSG content was calculated
comparing ΔA412 in the samples to a standard GSSG
curve (0–1 μM). The GSSG/2GSH ratio was calcu-
lated using GSSG and GSH-Eq measurements and
was expressed as GSSG:GSH-Eq. Results were ex-
pressed in nmol of GSH-Eq, GSH or GSSG per g of
wet tissue.
2.4. Enzyme activities
Previous to enzyme activity determinations, each
tissue sample was homogenized in 1:20 w/v phosphate
buffer (50 mM, pH 7.5; EDTA 1 mM; PMSF 1 mM) and
centrifuged at 2000 ×g for 20 min at 4 °C. The super-
natant was taken and used immediately for the analysis
(Vázquez-Medina et al., 2006).
2.4.1. Glutathione disulfide reductase (EC 1.6.4.2)
Glutathione disulfide reductase (GR) activity was as-
sayed by following the oxidation of NADPH by GSSG
in potassium phosphate buffer. KPi buffer (500 mM),
EDTA (50 mM), NADPH (2 mM), deionized water,
50 μL of sample and GSSG (10 mM) were mixed in a
cuvette. Change in absorbance per minute at 340 nm
(ΔA340) was recorded. Two blanks, one in absence of
sample and another one in absence of GSSG were run.
GR activity was calculated using the NADPH extinction
coefficient (6.22 M/L) and expressed in mU of GR per
mg of protein (Hermes-Lima and Storey, 1995).
2.4.2. Glucose 6 phosphate dehydrogenase (EC 1.1.1.49)
Glucose-6-phosphate dehydrogenase (G6PDH) ac-
tivity was measured by following NADP+ reduction in
a mixture containing KPi Buffer (500 mM), MgSO4
(5 mM), NADP+ (0.2 mM), glucose-6-phosphate (G6P,
2 mM) and sample (Lushchak et al., 2001). ΔA340 was
recorded and G6PDH activity calculated and expressed
in mU of G6PDH per mg of protein.
2.5. Oxidative damage
2.5.1. Thiobarbituric acid reactive substances assay
The amount of lipid peroxidation was assessed, as
previously described, by determining the tissue con-
tent of thiobarbituric acid reactive substances (TBARS)
(Ohkawa et al., 1979; Persky et al., 2000; Zenteno-Savin
et al., 2002). A standard curve of malondialdehyde bis-
(diethylacetal) was run in parallel with the samples, and
the concentration of TBARS in the samples was cal-
culated from this standard curve. Results were expressed
in nmol of TBARS per g of wet tissue.
2.5.2. Carbonyl proteins
Oxidative damage to proteins was quantified as car-
bonyl protein content (Levine et al., 1994). Frozen
samples were homogenized (1:20 w/v) in ice-cold
5% w/v sulfosalicylic acid and then centrifuged at

Table 1
Glutathione levels (nmol/g of wet tissue) in ringed seal (n = 11) and domestic pig (n = 10) heart, kidney, liver, lung and muscle
Species Tissue GSH-Eq (GSH + 2GSSG) GSH (reduced) GSSG (oxidized) GSSG:GSH-Eq
Ringed seal Heart 2918.14 ± 227.23a 2621.40 ± 241.48a 148.36 ± 10.19a 0.0578 ± 0.009b
Kidney 1121.79 ± 19.15c 1071.38 ± 20.74b 25.20 ± 2.59c 0.0226 ± 0.002b
Liver 327.24 ± 4.28b 266.52 ± 4.18c 30.4 ± 0.29c 0.0928 ± 0.001b
Lung 830.24 ± 5.69c 822.16 ± 5.69b 4.04 ± 0.06b 0.0048 ± 0.00b
Muscle 742.47 ± 67.54c 369.54 ± 37.62c 186.46 ± 21.64a 0.5424 ± 0.06a
Domestic pig Heart 117.47 ± 0.04⁎ 73.57 ± 4.545⁎ 21.95 ± 2.28⁎ 0.133 ± 0.01⁎
Kidney 490.33 ± 142.02⁎ 473.25 ± 142.26⁎ 8.54 ± 0.31⁎ 0.022 ± 0.002
Liver 451.21 ± 33.33 443.85 ± 33.11 3.68 ± .31⁎ 0.008 ± 0.001⁎
Lung 262.69 ± 65.08⁎ 248.31 ± 68.02⁎ 7.18 ± 1.64 0.060 ± 0.021⁎
Muscle 124.47 ± 7.22⁎ 66.81 ± 5.895⁎ 28.83 ± 2.74⁎ 0.157 ± 0.008⁎
Results are presented as mean ± SEM. Different letters denote significant differences among tissues (for ringed seal only); ⁎ denote significant
differences between species, p b 0.05. GSH-Eq = total glutathione (GSH + 2GSSG), GSH = glutathione, GSSG = glutathione disulfide.
 J.P. Vázquez-Medina et al. / Journal of Experimental Marine Biology and Ecology 345 (2007) 110–118 113

HCl (2 M) and were run in parallel with the samples.

Maximum absorbance in the range of 360–410 nm was
recorded and the final carbonyl protein values were
expressed, using the extinction coefficient of 22 (mM)
(L)− 1, in nmol of carbonyl proteins per g of wet tissue.

2.6. Total proteins

Soluble protein content in tissue homogenates was

measured following the method of Bradford (Bradford,
1976) using the Bio-Rad© prepared reagent and bovine
serum albumin as standard.

Fig. 1. A) Total glutathione content (GSH-Eq: GSH + 2GSSG) an

B) GSSG:GSH-Eq ratio in domestic pig ( n = 10) and ringed seal
(■ n = 11) tissues. Results are expressed in nmol/g wet tissue and
are presented as mean ± SEM. ⁎ = differences between species,
p b 0.05.

19,000 ×g at 4 °C for 5 min. Supernatant was removed

and 0.5 mL of 2,4-dinitrophenyl-hydrazine solution
(DNPH 10 mM in 2 M HCl) was added to the pellet.
Samples were kept at room temperature for 1 h (tubes
were vortexed during 40 s every 15 min). Then, 0.5 mL
of 20% TCA was added and the tubes were centrifuged
at 15,000 rpm for 3 min. Supernatants were again
discarded and the excess of DNPH was removed by
washing the pellet three times with 1:1 ethanol: ethyl
acetate and by centrifugation for 5 min at 15,000 rpm at
room temperature. The pellet was dissolved in guani- Fig. 2. A) Glutathione disulphide reductase (GR) and B) glucose-6-
phosphate dehydrogenase activities in domestic pig ( n = 10) and
dine chloride (6 M) and incubated for 15 min at 37 °C. ringed seal (■ n = 11) tissues. Results are expressed in mU/mg protein,
Then, samples were centrifuged at 15,000 rpm for and are presented as mean ± SEM. ⁎ = differences between species,
5 min. Blanks were prepared by replacing DNPH with p b 0.05.
114 J.P. Vázquez-Medina et al. / Journal of Experimental Marine Biology and Ecology 345 (2007) 110–118

Table 2
Activities of glutathione disulfide reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH) (mU/mg of protein), and markers of oxidative
damage to lipids and proteins (nmol/g wet tissue) in ringed seal heart, kidney, liver, lung and muscle (n = 11)
Tissue GR G6PDH TBARS Carbonyl proteins
a a
Heart 7.52 ± 2.52 15.56 ± 3.551 0.252 ± 0.02 85.35 ± 21.18a
Kidney 5.80 ± 1.40 121.32 ± 42.72ab 0.107 ± 0.01c 128.22 ± 12.33a
Liver 3.97 ± 0.67 134.47 ± 27.96b 0.672 ± 0.08b 63.72 ± 9.49a
Lung 6.65 ± 2.11 67.64 ± 17.85ab 0.234 ± 0.00c 281.53 ± 40.59b
Muscle 4.65 ± 1.20 71.78 ± 29.19ab 0.330 ± 0.05c 369.50 ± 31.86b
Results are presented as mean ± SEM. Different letters denote significant differences among tissues, p b 0.05. TBARS = thiobarbituric acid reactive
substances.

2.7. Statistical analysis activity among ringed seal tissues was observed in liver
( p b 0.05) (Fig. 2, Table 2). Ringed seal heart had lower
Results are reported as mean ± SEM. All data were G6PDH activity than liver ( p = 0.03) (Table 2).
subjected to normality and homocedasticity tests. Sig-
nificant differences between tissues (for ringed seal
only) and species were estimated using Krustal–Wallis
and Mann–Whitney tests (Zar, 1999). Statistical
significance was accepted when p b 0.05. Statistical
analyses were performed using the SYSTAT© 11.0
(SPSS, Richmond, CA, USA) software.

3. Results

3.1. Glutathione assay

Results of GSH-Eq, GSH, GSSG and GSSG:GSH-

Eq determinations are summarized in Table 1 and Fig. 1.
GSH-Eq content was higher in seal than in pig heart,
kidney, lung and muscle ( p b 0.05) and presented no
differences between species in liver (Fig. 1). All ringed
seal tissues, except liver, had higher ( p b 0.05) GSH
content than pig tissues; ringed seal heart had higher
( p b 0.05) GSH content than ringed seal kidney or lung,
and liver or muscle (Table 1). Heart had the highest
GSH-Eq levels among all the seal tissues ( p b 0.05)
while liver had the lowest GSH-Eq content ( p b 0.05)
(Table 1). GSSG content was higher ( p b 0.05) in ringed
seal than in pig heart, kidney, liver and muscle (Table 1).
GSSG:GSH-Eq ratio was higher in pig than in ringed
seal heart and lung ( p b 0.05); was higher in ringed seal
than in pig liver and muscle ( p b 0.05) and had no
difference in kidney (Fig. 1). The highest GSSG:GSH-
Eq ratio among ringed seal tissues was observed in
muscle ( p = 0.003) (Table 1).

3.2. Enzyme activities

GR and G6PDH activities were significantly higher in

Fig. 3. A) TBARS and B) carbonyl proteins content (nmol/g wet
all ringed seal than in pig tissues ( p b 0.05) (Fig. 2). No tissue) in domestic pig ( n = 10) and ringed seal (■ n = 11) tissues.
significant differences were shown in GR activities Results are presented as mean ± SEM. ⁎ = differences between species,
among ringed seal tissues (Table 2). The highest G6PDH p b 0.05.
 J.P. Vázquez-Medina et al. / Journal of Experimental Marine Biology and Ecology 345 (2007) 110–118
3.3. Oxidative damage
TBARS content was higher in pig than in ringed seal
heart and lung, and lower in pig than in ringed seal
muscle ( p b 0.05) (Fig. 3). The highest TBARS content
in ringed seal tissues was observed in liver and the
lowest in kidney ( p b 0.05) (Fig. 3, Table 2). Higher
carbonyl protein content was present in pig than in
ringed seal heart, kidney, liver and muscle ( p b 0.05)
(Fig. 3). Ringed seal lung and muscle had higher protein
carbonyl content than the other ringed seal tissues
( p b 0.05) (Table 2).
4. Discussion
The rate of utilization of GSH in cellular oxidation/
reduction reactions, the enzymatic reduction of hydro-
peroxides, xenobiotic metabolism and regeneration of
reduced forms of redox pairs are factors that modulate
the amount of GSH levels in mammalian cells (Kirlin
et al., 1999; Schafer et al., 2000). Higher content of
GSH-Eq, GSH and GSSG (Table 1) and higher activities
of GR and G6PDH (Fig. 2) in ringed seal than in pig
heart, kidney, lung and muscle suggest that the recycling
and maintenance of GSH in seal tissues occurs at a high
rate, and support the hypothesis that the permanent
availability and the active participation of GSH (as a
substrate for enzymes such as GPx or GST) contribute
importantly in scavenging ROS produced in response to
diving in ringed seal tissues (Vazquez-Medina et al.,
2006).
Previous studies (Zenteno-Savin and Elsner, 1998,
U
2000; Zenteno-Savin et al., 2002) suggest that O2−
production is higher in ringed seal than in pig heart,
kidney and muscle as a direct consequence of the
ischemia/reperfusion associated with the diving re-
sponse (Elsner, 1999); however, oxidative damage to
U
lipids and proteins is not related to this high O2−
production (Fig. 3). Apparently, SOD, CAT, GPx and
GST activities participate actively in the antioxidant
protection in ringed seal heart, liver, lung and muscle,
but not in kidney (Elsner et al., 1998; Vazquez-Medina
et al., 2006).
The results of the present study support the idea that
low-weight non-enzymatic antioxidants such as GSH
have an important role in ROS scavenging in seal
kidney. Seal kidney is highly tolerant of ischemia
(Halasz et al., 1974; Behrisch and Elsner, 1980). During
ischemia seal kidney accumulates hypoxanthine (HX)
(Elsner et al., 1995, 1998) and during reperfusion it
U U
produces O2− at high rates. Apparently, this O2− pro-
duction in ringed seal kidney is compensated for by an
115
elevated antioxidant capacity (Zenteno-Savin et al.,
U
2002). Elevated rates of mitochondrial O2− generation
are counteracted by elevated levels of GSH (Halliwell
and Gutterdige, 1999). During ischemia in in vivo
models there is an increase in the supply of GSH pre-
cursors, a stimulus to activate GSH synthase and sup-
pression of lipid peroxidation (Kamencic et al., 2001).
Other findings that support the previous idea are
those of Murphy and Hochachka (1981), who reported
an increase in GSH circulating concentration in seals
after forced dives, and of Wilhem-Filho et al. (2002),
who reported higher GSH concentrations in the blood of
marine mammals than in the blood of terrestrial mam-
mals. High GSH levels have been also observed in other
animal tissues that are exposed to changes in oxygen
availability by hibernation, estivation, desiccation, envi-
ronmental hypoxia or diving (Hermes-Lima and Zen-
teno-Savin, 2002; Ramos-Vasconcelos and Hermes-
Lima, 2003; Bickler, 2004; Ramos-Vasconcelos et al.,
2005; Willmore and Storey, 2005).
Intracellular redox homeostasis is crucial for gene ex-
pression, enzyme activation and control of ROS levels
(Arrigo, 1999). Higher GSSG:GSH-Eq ratio in ringed
seal than in pig muscle is probably related to the higher
U
O2− production in ringed seal than in pig muscle reported
by Zenteno-Savin et al. (2002), and is reflected in higher
TBARS content but not in higher protein carbonyl levels
in this tissue (Fig. 3), suggesting an acute antioxidant
U
protection of GSH to remove HO and singlet oxygen,
thus preventing oxidative damage to proteins and bal-
ancing redox status (Arrigo, 1999). GSSG:GSH-Eq ratio
was also higher in ringed seal than in pig liver but was not
reflected in a higher oxidative damage to lipids or proteins
(Fig. 3). An accumulation of oxidized metabolites was
observed in rainbow trout liver after starvation (Barroso
et al., 1998). It is possible that diving seal liver presents
a similar pattern reflected in the GSSG:GSH-Eq ratio. On
the other hand, it is also possible that the second step of
the detoxification processes in liver, which involves GST
participation (Tsuchida and Sato, 1992; Kirlin et al.,
1999), promotes the accumulation of GSH in pig hepatic
tissue to support a higher activity of this enzyme in pig
than in ringed seal (Vázquez-Medina et al., 2006). Pig
liver was also the only tissue in this species that presented
higher (albeit non-significant) GSH and GSH-Eq con-
centrations (Table 1; Fig. 1), this is probably reflected in
the lower GSSG:GSH-Eq ratio.
Higher GR and G6PDH activities in all ringed seal
than in pig tissues (Fig. 2) probably contribute to sustain
redox homeostasis. GR is an enzyme that plays an im-
portant role in maintaining glutathione in its reduced
form recycling GSSG to GSH at the expense of NADPH
116 J.P. Vázquez-Medina et al. / Journal of Experimental Marine Biology and Ecology 345 (2007) 110–118
(Fujii et al., 2000). G6PDH has been identified as a
crucial enzyme that recycles NADP+ to NADPH,
completing the GSH cycling metabolism, and inhibits
H2O2-induced cell death by affecting the redox potential
to maintain GSH levels (Tian et al., 1999; Leopold and
Loscalzo, 2000). Increases in GR and G6PDH activities
have been implicated in the antioxidant defense re-
sponse against reoxygenation-derived ROS production
in animals adapted to drastic chances in oxygen avail-
ability (Lushchak et al., 2001; Hermes-Lima and
Zenteno-Savin, 2002).
Differential ischemia/reperfusion in ringed seal tis-
sues during and after diving (Elsner et al., 1966; Gooden
and Elsner, 1985) results in differences in post-diving
ROS production (Zenteno-Savin and Elsner, 2000),
antioxidant enzyme response (Vazquez-Medina et al.,
2006) and GSH levels (Table 1). During a dive, seal
heart receives intermittent blood flow such that oxygen
consumption decreases while maintaining reduced
cardiac function (Gooden and Elsner, 1985; Elsner,
1999). These intermittent reductions of blood flow seem
to be a natural protective mechanism of ringed seal heart
that induces the activities of SOD, GST and GPx (Elsner
et al., 1998; Vazquez-Medina et al., 2006), the activities
of GR and G6PDH (Fig. 2), an increase in GSH and
GSH-Eq levels (Table 1, Fig. 1), and other mechanisms
that are involved in oxygen sensing in seal tissues, such
as the accumulation and stabilization of hypoxia induc-
ible factor 1α (HIF-1α) (Johnson et al., 2004, 2005), a
key protein that coordinates the adaptative homeostatic
responses to hypoxia by regulating the expression of
vascular, glycolytic and cell cycle regulatory genes
(Bunn and Poyton, 1996), and that is probably
regulating the expression of heat shock factor (Hsf),
the main switch in heat shock proteins (Hsps) activation
and a critical element in the adaptation to reoxygena-
tion-derived ROS production (Date et al., 2005; Baird
et al., 2006).
In terrestrial models, GSH dietary supplementation
protects heart against adverse consequences of ische-
mia/reperfusion (Ahmad et al., 2001; Ramires and Ji,
2001). In other studies GSH levels appear to be the
cellular control mechanism that regulates GPx activity
and H2O2 levels in response to changes in oxygen ten-
sion in coronary arteries (Mohazzab-H et al., 1999), and
the mechanism that controls cellular PO2 (Del Corso
et al., 2002).
Other studies (Haddad, 2002a,b,c; Haddad and Harb,
2005) suggest that GSH-associated metabolism has a
crucial role in controlling the non-hypoxic activation of
HIF-1α, a phenomenon uncoupled from the classical
hypoxic induction of this protein (Richard et al., 2000).
A reducing cell environment seems to be necessary
for HIF-1 stabilization (Salceda and Caro, 1997). In
the North Sea eelpout heat or cold-induced hypoxia
(which seems to have similar implications as ischemia/
reperfusion in mammals) favors a more reduced cellular
redox state and high GSH levels that activate HIF-1
DNA binding previous to reoxygenation (Heise et al.,
2006a,b).
Maintaining high GSH levels in preparation for post-
diving ROS production (Hermes-Lima and Zenteno-
Savin, 2002) seems to be an important mechanism for
counteracting dive-associated ischemia/reperfusion be-
cause production of GSH is quick and less expensive
than enzyme synthesis (Kosower and Kosower, 1978;
Meister and Anderson, 1983) and probably promotes a
highly reduced redox status that induces the redox-
dependent activation (Huang et al., 1996; Ema et al.,
1999; Lando et al., 2000) of HIF-1 genes that exist in the
ringed seal genome (Johnson et al., 2004) and are ex-
pressed constitutively in ringed seal heart, kidney, lung
and muscle (Johnson et al., 2005).
Another advantage of maintaining high levels of
GSH is the scavenging capacity of this thiol against
U
peroxynitrite that is formed by the reaction of the O2−
produced post-diving (Zenteno-Savin et al., 2002) with
nitric oxide (NO), and suppresses HIF-1 stabilization
and DNA binding activity (Agani et al., 2002).
In summary, the present study showed that GSH
levels and the activities of the enzymes implicated in its
recycling are actively involved in antioxidant protection
against dive-related ischemia reperfusion, ameliorating
protein and lipid oxidative damage, and maintaining a
highly reduced redox status. It is possible that the latter
controls the activation of other protecting mechanisms
that contribute to keep oxygen homeostasis response
during reoxygenation such as HIF-1, which probably
promotes the expression of HSPs that could be involved
in the preparation for post-diving ROS production in
ringed seal tissues.
Acknowledgements
Members of the North Slope Borough, the Alaska
Department of Wildlife Management, the Barrow Arc-
tic Science Consortium and Inupiat Eskimo hunters
provided logistic support and assistance in obtaining
ringed seal samples. Sampling was performed under
terms of a marine mammal scientific permit issued to RE
by the Office of Protected Species, US National Marine
Fisheries Service. Samples were imported to Mexico
under terms of import permits issued to TZS by Instituto
Nacional de Ecología. Dr. José María Acosta and
 J.P. Vázquez-Medina et al. / Journal of Experimental Marine Biology and Ecology 345 (2007) 110–118
personnel of Rastro Municipal de La Paz assisted in
obtaining pig samples. Norma Olguín (CIBNOR)
helped in sampling extractions. Gabriela Ramos, Daniel
Prado, Orlando Furtado and Dr. Marcelo Hermes-Lima
(UBr) provided instruction and advice on analytic
techniques. This research was funded by grants from
the Office for Naval Research, UC-Mexus, SEMAR-
NAT-CONACYT, UAF, and CIBNOR. JPVM is a
recipient of a CONACYT scholarship (200974). [SS]
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